Biochemical analysis of tumor rejection antigen and its role of oncogenesis.

对RL*1特异性CTL识别的抗原肽进行反相高效液相色谱直接测序。分子分析表明，抗原肽是通过插入小鼠白血病病毒LTR，从原癌基因c-akt的5'-未翻译区获得的，并含有6个未知来源的核苷酸。在本研究中，我们研究了这种改变的Akt分子及其在RL*1.1肿瘤发生中的作用。利用兔抗c-Akt合成多肽抗体进行Western blot分析，发现RL*1细胞中除了56 KD的正常c-Akt蛋白外，还有一个59 KD的蛋白。2. 59 KD改变的Akt分子（RL-Akt）表达量约为正常c-Akt的10倍，在细胞膜部分和细胞质部分均检测到，而c-Akt仅在细胞质部分检测到。3. RL-akt转基因NIH 3T3细胞生长良好，4月4日形成菌落。建立RL-Akt缺失突变体和RL-Akt弱表达突变体。5. 为了评估RL- akt对体内生长速度的影响，将这些突变体和亲代RL*1细胞分别移植到BALB/c小鼠中，亲代RL*1细胞生长迅速，但弱表达突变体生长缓慢，缺失突变体生长更慢。这些结果表明，RL- akt在膜内的过表达和定位导致了生长因子- pi3通路的有效信号转导，并参与了RL*1白血病的恶性过程。关于RL-Akt对宿主免疫系统影响的研究正在进行中。

The antigen peptides recognized by RL*1 specific CTL were determined by direct sequencing of the fraction separated by reversed-phase high performance liquid chromatography. Molecular analysis revealed the antigen peptide were derived from the 5'-untransrated region of proto-oncogene c-akt by insertion of a murine leukemia virus LTR together with six nucleotides of unknown origin. In this study, we investigated this altered Akt molecule and its role of oncogenesis in RL*1.1. Western blot analysis using plyclonal rabbit anti c-Akt synthetic polypeptide antibodies revealed a protein 59 KD in addition to 56 KD normal c-Akt protein in RL*1 cells. 2. The 59 KD altered Akt molecule (RL-Akt) was expressed at about ten times level to normal c-Akt and was detected in the membrane fraction and in the cytosolic fraction, although the c-Akt was detected only in the cytosolic fraction. 3. RL-akt transgeneic NIH 3T3 cells grew well, forming colonies in soft apr. 4. RL-Akt deletion mutant and RL-Akt weakly expressed mutant were established. 5. To assess the effect of RL-Akt in the growth rate in vivo, these mutants and parental RL*1 cells transplanted in BALB/c mice, respectively, parental RL*1 cells grow rapidlly, but weakly expressed mutant grow slowly, and deletion mutant did more slowly.These observations suggests that the over expression and localization in the membrane of RL-Akt resulted in effective signal transduction of growth factor-PI3 pathway and these participates in malignancy in RL*1 leukemia. The study of the effects of RL-Akt to the host immune system are going now.